More than 100 patients with thoracic malignancies have been treated on a series of clinical protocols examining toxicities and clinical responses following infusions of DNA demethylating agents (Decitabine; DAC), and HDAC inhibitors, such as romidepsin (DP) alone or in combination with other investigational agents. Collectively these trials demonstrated no objective clinical regressions, although prolonged stabilization of disease (4 - 12months) was observed in approximately 10% of patients. Nearly one quarter of all patients receiving DAC infusions exhibited increased expression of p16, MAGE-3, or NY-ESO-1 in tumor tissues. Serologic responses to NY-ESO-1 were observed in several patients receiving DAC for more than six months. Approximately 50% of patients receiving DP infusions exhibited increased intratumoral levels of H3Ac and p21. In addition, several patients exhibited enhanced expression of NY-ESO-1 and MAGE-A3 in tumor biopsies following DP infusions. Micro-array analysis of laser captured tumor cells from pre and post treatment biopsies from patients receiving DAC, DP or sequential DAC/DP infusions revealed a shift from a lung cancer gene signature to one observed in normal respiratory epithelia. These early trials provided proof of concept for the use of epigenetic regimens in combination with immunologic interventions for the treatment of thoracic malignancies. Because CT-X antigens appear to be preferentially expressed in pluripotent tumor cells, it is conceivable that autologous epigenetically modified tumor cells may be unique, personalized vaccines to induce immune responses to cancer stem cells. Difficulties regarding reliable establishment of primary cell lines limited our ability to formally evaluate this issue in the clinic, and these protocols were closed. In an attempt to circumvent these problems, we have examined if vaccines produced from allogeneic cancer cells can induce broad immunity to CT-X antigens that potentially can be up-regulated in thoracic malignancies by gene induction regimens. In a recent phase II trial, we have evaluated the immunogenicity of a freeze thaw lysate of H1299 lung cancer cells. This NSCLC line was established at the NCI and exhibits high level expression of numerous relevant CT-X gene expression relative to K562-GM due to amplification of the X chromosome. In an ongoing first-in- humans trial, 21 patients with thoracic malignancies rendered NED by conventional therapy were randomized to receive freeze-thaw lysates from H1299 lung cancer cells exhibiting high-level CTA expression with Iscomatrix via intradermal injection q month x 6 +/- oral metronomic cy/cel. The primary endpoint was serologic response to purified tumor antigens one month after the 6th vaccination. All patients exhibited local and systemic inflammatory responses lasting 72-96 hours following vaccinations. There were no treatment related toxicities. 14 patients (67%) completed all six vaccinations; 7 patients were removed from study early due to disease recurrence. 8 patients (57%) exhibited serologic responses to NY-ESO-1. Additional responses were observed against GAGE7, XAGE and MAGE-C2. Vaccine therapy also decreased percent Tregs (p=0.0067) and PD-1 expression on Tregs (p=0.0023), as well as PD-L1 expression on CD14+ monocytes (p=0.0089), classical monocytes (p=0.0159), and intermediate monocytes (p= 0.0031). Cy/cel did not increase immune responses or enhance vaccine-induced alterations in peripheral immune subsets.H1299 lysate vaccines induce immune responses to CTAs, and modulate peripheral immune subsets in a manner that may enhance antitumor immunity. These findings support evaluation of this lysate in combination with immune checkpoint inhibitors in thoracic oncology patients. Results of this trial have been selected for oral presentation at the American College of Surgeons Annual Clinical Congress, and a manuscript pertaining to these findings will be submitted for publication in the near future. A major limitation regarding the use of DNA demethylating agents in cancer immunotherapy regimens pertains to the short half-life (5min) and poor biodistribution due to cytidine deminase (CDA) which is present at high levels throughout the body. Recent studies in non-human primates and a clinical trial in patients with sickle cell disease, have demonstrated that an oral formulation of tetrahydrouridine (THU)- a compound that has been previously administered intravenously to hundreds of cancer patients with no toxicities can markedly increase Cmax (50nM)and T1/2 ( 4 hours) of oral Decitabine. These findings have provided the rationale for a phase II trial evaluation oral DAC/THU and the immune checkpoint inhibitor Nivolumab as second line therapy for patients with NSCLC, as well as a phase I/II dose escalation study of oral DAC/THU and pembrolizumab as first line therapy for patients with inoperable NSCLC exhibiting high level PDL1 expression. Comprehensive state of the art translational analyses of PD endpoints including multiplex quantitative IHC, multiparametric mass cytometry (CyTOF) and next-gen sequencing will be conducted in the Translational Imuno-Oncology Laboratory at Yale University. Additional studies including analysis of immune subsets, circulating tumor cells and methylation status of circulating tumor DNA will be performed at the NIH Clinical Center.